Cell-free replication system and uses thereof

Abstract

The invention relates to improvements in cell-free systems for initiating DNA replication under cell cycle control. The system comprises a synchronous population of G1 nuclei which have been released from G0; and S phase cytosol. In a preferred aspect, a polypeptide is supplied to the system, wherein the polypeptide is Cdc6 and/or at least one MCM protein. The system is suitable for DNA replication assays, for example to test substances which modulate DNA replication.

What is claimed is:  
     
       1. A method for conducting a cell-free assay for the initiation of DNA replication which comprises: 
       providing a synchronous population of G1 nuclei obtained from non-transformed cell lines which have been released from G0;  
       bringing said nuclei into contact with somatic cell S phase cytosol under conditions suitable for DNA replication to occur;  
       incubating a test substance with said nuclei and cytosol; and  
       determining whether or not initiation of DNA replication occurs in the presence of said test substance.  
     
     
       2. The method of  claim 1  which comprises supplying to the S phase cytocol at least one protein selected from cdc6 and an MCM protein. 
     
     
       3. The method of  claim 2 , further including permeabilising the harvested nuclei prior to bringing said nuclei into contact with Cdc6 and/or at least one MCM protein. 
     
     
       4. A method according to  claim 1 , wherein the substance is an inhibitor of DNA synthesis. 
     
     
       5. A method according to  claim 1 , wherein the substance is a stimulator of DNA synthesis. 
     
     
       6. The method of  claim 1  wherein when said test substance is identified as an agent which modulates DNA replication, said test substance is modified. 
     
     
       7. The method of  claim 6  wherein said test substance is modelled according to its physical properties including at least one physical property selected form the group of stereochemistry, bonding, size and charge, or said test substance is modelled by computational analysis. 
     
     
       8. A method according to  claim 6  wherein the substance is modified to alter a property of the substance. 
     
     
       9. A method according to  claim 8  wherein the substance is modified to alter its ability to modulate initiation of DNA replication. 
     
     
       10. A method of  claim 1  wherein when said test substance is identified as an agent which modulates DNA replication, said test substance is formulated into a composition including at least one additional component. 
     
     
       11. A method according to  claim 10  wherein the composition includes a pharmaceutically acceptable excipient.